Progress in understanding the mode of action of specific peptide inhibitors of pepsin has been hampered by a lack of information about the mechanism of the enzyme. This is due to an absence of a good, versatile pepsin substrate that can be used for mechanistic studies over a broad range of conditions. We have developed a new substrate that is 1) soluble over the whole pH range of interest, 2) specifically cleaved by pepsin at a defined bond, and 3) has fluorescent properties that allow the direct, continuous photometric assay. This substrate, and others designed by a combination of principles, will be used to study the mechanism of pepsin over a broad pH range (1-7). This will provide a foundation for deeper studies of the action of our extensive series of peptide inhibitors based on the zymogen activation fragments. We will also use fluorescence spectroscopy to study the interaction of specifically labeled peptide inhibitors with pepsin. Some of this work will involve the use of low temperature (0 to minus 60 degrees C) to slow down kinetic processes enough to permit observation. In particular, we propose to study the activation mechanism in more detail by using low temperatures to separate the conformational changes from covalent bond cleavages. We will also employ stopped flow kinetics where necessary to resolve kinetics steps. We will continue to probe the structural requirements for strong inhibition in the pepsinogen (1-16) sequence. Our attention will focus on shorter analogs with high hydrophobic potential based on our extensive preliminary findings. These studies are aimed at a fuller understanding of the mechanism of inhibition of pepsin and will permit the rational design of more effective structures that could be clinically effective.